Summary

Temporal and spatial coordination of multiple cell fate decisions is
essential for proper organogenesis. Here, we define gene interactions that
transform the neurogenic epithelium of the developing inner ear into
specialized mechanosensory receptors. By Cre-loxP fate mapping, we show that
vestibular sensory hair cells derive from a previously neurogenic region of
the inner ear. The related bHLH genes Ngn1 (Neurog1) and
Math1 (Atoh1) are required, respectively, for neural and
sensory epithelial development in this system. Our analysis of mouse mutants
indicates that a mutual antagonism between Ngn1 and Math1
regulates the transition from neurogenesis to sensory cell production during
ear development. Furthermore, we provide evidence that the transition to
sensory cell production involves distinct autoregulatory behaviors of
Ngn1 (negative) and Math1 (positive). We propose that
Ngn1, as well as promoting neurogenesis, maintains an uncommitted
progenitor cell population through Notch-mediated lateral inhibition, and
Math1 irreversibly commits these progenitors to a hair-cell fate.

Morphological and genetic homologies between the sensory organs of the
vertebrate ear and insects raise the question of whether neurons and hair
cells derive from a common progenitor cell type
(Fekete and Wu, 2002;
Adam et al., 1998). Direct
evidence for this is limited to one recent study in chicken that found clonal
relatives of VIIIth ganglion neurons within the utricular macula and adjacent
non-sensory epithelium (Satoh and Fekete,
2005). Existing evidence for such a relationship in mammals is
circumstantial (Matei et al.,
2005; Raft et al.,
2004).

Otic neurogenesis and hair cell generation are each dependent on an
Atonal-related bHLH transcription factor. neurogenin 1 (Ngn1; also
known as Neurog1 - Mouse Genome Informatics) is necessary for the
commitment of otocyst epithelial cells to a neural fate, as
Ngn1-/- embryos lack an VIIIth cranial ganglion and fail
to express neural fate markers in the otocyst
(Ma et al., 1998;
Ma et al., 2000). By contrast,
Math1 (also known as Atoh1 - Mouse Genome Informatics) is
necessary and sufficient for hair cell generation
(Bermingham et al., 1999;
Zheng and Gao, 2000;
Izumikawa et al., 2005). Thus,
during ear development, Ngn1 and Math1 function as
determination factors, but it is not known whether their expression is
regulated in a coordinated manner. In other neural systems, bHLH genes
cross-regulate to control the commitment of progenitor cells to alternative
fates (Bertrand et al., 2002).
Examples include the involvement of Mash/Math/Ngn1 genes in the
sequential production of retinal neurons and glia
(Inoue et al., 2002;
Akagi et al., 2004), and in the
simultaneous production of distinct neural subtypes in the forebrain and
spinal cord (Fode et al.,
2000; Gowan et al.,
2001).

Here, we identify a progenitor cell field that produces both neurons and
hair cells and describe genetic interactions mediating a neural-hair cell fate
decision. We show that neural precursor and hair cell production overlap in
the otic epithelium for several days and demonstrate by fate mapping that the
Ngn1-expressing neurogenic region is transformed into the sensory
maculae of the utricle and saccule. We propose that this transformation is
governed by a mutual antagonism between Ngn1 and Math1. We
also show that Ngn1 negatively regulates its own expression through
Notch-mediated lateral inhibition, whereas Math1 positively regulates
its own expression. Differential autoregulation of Ngn1 and
Math1 provides an explanation for the progression toward sensory
epithelial development over time. Ngn1, as well as generating neural
precursors, functions via lateral inhibition to maintain an uncommitted
progenitor cell population for sensory epithelial development; Math1,
in turn, irreversibly commits these progenitors to a hair cell fate.

RESULTS

Neurogenesis and hair cell production overlap spatially and
temporally in the developing utricle and saccule

We performed RNA in situ hybridization for NeuroD and
Math1 to characterize any temporal and spatial overlap between
neurogenesis and hair cell production (Fig.
1A) (Fritzsch et al.,
2002). NeuroD (also known as Neurod1 - Mouse
Genome Informatics), a neural differentiation gene
(Bertrand et al., 2002), is
expressed under the control of Ngn1 in the otic epithelium
(Ma et al., 1998) and marks
cells that are committed to the neural lineage. We also hybridized a
NeuroD RNA probe to tissue from a Math1-GFP transgenic reporter mouse
(Lumpkin et al., 2003).
Math1 is the earliest known specific marker of hair cells
(Bermingham et al., 1999).

The number of NeuroD+ cells within the epithelium
increases between E9 and E10.5 (Raft et
al., 2004) (Fig.
1A). Between E11.5 and E12.5, NeuroD expression split
into two distinct regions of neurogenesis that would ultimately lie within the
developing utricle and saccule (Fig.
1B,C, cyan). Neurogenesis declined from E11.5 onward
(Fig. 1D,E), but a few
delaminating NeuroD+ cells were still present in the
utricle as late as E17.5 (5±4 cells, n=3 ears; data not
shown). A comparison of NeuroD and Ngn1 expression in the
otic epithelium revealed no differences in their patterning (see Fig.
S1A,D,E,H in the supplementary material).

During the decline in neurogenesis, Math1 mRNA expression begins
and is maintained in all hair cells through to at least E17.5
(Shailam et al., 1999). At
E11.5, two Math1+ stripes appeared within the
NeuroD domain along its lateral and medial borders
(Fig. 1B,F,G). Between E11.5
and E12.5, Math1-GFP+ cells in these stripes increased in number by
8- to 10-fold and formed the nascent maculae of the utricle and saccule.
Initially, Math1-GFP+ and NeuroD+ cells
intermingled (Fig. 1H,I), but
later lay on either side of a border that delineates the macula and its
adjacent neurogenic domain (Fig.
1J). Math1 expression associated with the cristae first
appeared as separate foci outside the NeuroD+ domain at
around E12 (data not shown). We found no temporal overlap of Math1
and NeuroD expression in the cochlea (data not shown), suggesting
that neurogenesis and hair cell generation do not coincide in the auditory
end-organ. Taken together, our data reveal that neurogenesis is maintained
through stages of hair cell production in the utricle and saccule, but
declines sharply as Math1 expression and hair cell production
increase.

Maculae of the utricle and saccule derive from the
Ngn1-expressing domain of the otocyst

Our expression analysis raised the possibility that sensory maculae of the
utricle and saccule, but not the cristae or organ of Corti, derive from the
neurogenic region of the otocyst. To test this, we permanently labeled cells
of the neurogenic region using a BAC transgenic mouse line (Ngn1-CreER)
(E.J.K. and L.V.G., unpublished) that expresses a tamoxifen-inducible form of
Cre recombinase (CreER) under the control of Ngn1 regulatory elements
(see Fig. S1B,F in the supplementary material). This allowed us to identify
cells transiently expressing Ngn1 and their progeny after sensory
epithelia have formed. As expected, when the Ngn-CreER mouse was crossed with
the Z/EG reporter line (Novak et al.,
2000) and tamoxifen administered, roughly 50% of VIIIth cranial
ganglion neurons were permanently labeled in double-transgenic embryos
(Fig. 2A; see Fig. S1I in the
supplementary material). Importantly, we also found Ngn1 derivatives to be
present in sensory and non-sensory inner ear epithelia of embryos that had
been sacrificed after neurogenesis was largely complete
(Fig. 2B-G).

To follow the fate of Ngn1-expressing cells in the ear, we
administered tamoxifen twice daily from E8.5 until E13.5 to pregnant females
of Ngn1-CreER × Z/EG matings. We analyzed 20 double-transgenic right
ears from seven litters ranging in age from E13.5-16.5 and identified over
5000 labeled epithelial cells. Sensory epithelia were identified by the
presence of myosin VIIa protein, and the resulting distribution pattern of
epithelial Ngn1 derivatives is summarized in
Fig. 2G. Supporting the
hypothesis that maculae are the only sensory epithelia to derive from
neurogenic epithelium, Ngn1 derivatives were present in the utricular and
saccular maculae of all specimens analyzed
(Fig. 2B,C). For embryos
sacrificed at E14.5, tamoxifen administration from E8.5-13.5 yielded an
average of 157±25 Ngn1 derivatives per utricular macula (n=6
ears from two litters). We found a lower occurrence of such cells in the
saccular macula (82±24; n=6 ears from two litters). By
contrast, only one ear out of the 20 analyzed had Ngn1 derivatives in the
lateral crista (eight labeled supporting cells), and no such cells were
detected in the other cristae in our cohort of specimens. No Ngn1 derivatives
were detected in the organ of Corti. We were able to classify macular Ngn1
derivatives as differentiated myosin VIIa+ hair cells
(Fig. 2H), undifferentiated
myosin VIIa+ epithelial cells migrating within the apical-basal
plane of the epithelium (Fig.
2I), or as myosin VIIa- pseudostratified epithelial
cells (Fig. 2J). By E16.5, many
of these myosin VIIa- Ngn1 derivatives exhibited morphological
features of supporting cells (Fig.
2K, arrowheads).

Regions of non-sensory epithelium flanking the maculae of E13.5-16.5 ears
also contain labeled cells (Fig.
2D,E), but we found no Ngn1 derivatives in the semicircular
canals. In the auditory portion of the ear, Ngn1 derivatives were detected in
82% (14/17) of the cochleae analyzed (E13-16.5), but showed extreme
variability in their numbers (93±146 cells, n=8 cochleae at
E14.5). These cells commonly occupied the greater epithelial ridge (GER), a
non-sensory region of the cochlea that is adjacent to the organ of Corti
(Fig. 2F). However, as
described above, no Ngn1 derivatives were detected in the organ of Corti
itself.

Administration of tamoxifen only at placode/otocyst stages (E8.5 and E9.5)
resulted in the same spatial distribution of Ngn1 derivatives as described
above. Together, our results indicate that the utricular and saccular maculae,
as well as some non-sensory epithelium flanking these structures, derive from
the neurogenic region of the otocyst, and that Ngn1-expressing
otocyst cells or their descendants can differentiate as hair cells, supporting
cells, or as structural epithelial cells.

The Ngn1 domain contracts gradually and stereotypically in
the primordia of the utricle and saccule

To understand how the sensory maculae and their surrounding tissue arise
from neurogenic tissue, we analyzed changes in Ngn1 expression over
time using two different transgenic lines. A series of Ngn1-CreER × Z/EG
litters received initial tamoxifen exposures at progressively later
developmental time points from E8.5 onwards; once begun, tamoxifen
administration was continued twice per day until E13.5 and all litters were
sacrificed on E14.5. By quantifying GFP+ cells in the utricle of
these embryos, we confirmed that starting tamoxifen administration at
progressively later times leads to diminishing numbers of labeled Ngn1
derivatives (Fig. 3C). From
these experiments, we mapped the distributions of Ngn1 derivatives in the
utricular macula and its flanking non-sensory tissue
(Fig. 3E-E‴). The
distribution of cells actively expressing Ngn1 at E14.5 was obtained
from a different BAC transgenic line (Ngn1-GFP) that reports directly on
Ngn1 promoter activity (see Fig. S1C,G,J in the supplementary
material).

Our results for the utricle are consistent with a stereotyped reduction in
the area of Ngn1 expression over time. At E14.5, the active
neurogenic domain of the utricle, defined by expression of the Ngn1-GFP
reporter, lay medial to and centered along the anteroposterior axis of the
macula (Fig. 3A,B). When
tamoxifen was administered to Ngn1-CreER;Z/EG litters from E8.5-13.5
(Fig. 3E), Ngn1 derivatives
were present throughout the neurogenic domain (white region), the macular
sensory epithelium (gray region, defined by myosin VIIa expression), lateral
non-sensory tissue (between the utriclar macula and lateral crista, yellow
region), and posterior non-sensory tissue (between the utricular and saccular
maculae, yellow region, bottom). When tamoxifen was administered from E10.5 or
E11.5 to E13.5, Ngn1 derivatives were present at all these sites, except for
posterior non-sensory tissue (between the maculae,
Fig. 3E′). Finally, when
tamoxifen was administered from E12.5-13.5, Ngn1 derivatives were present only
within the neurogenic domain and a portion of the macular epithelium nearest
its border with the neurogenic domain (Fig.
3E″). Contraction of the Ngn1 expression domain is
therefore directional, occurring largely from lateral to medial in the
utricle. We observed similar Ngn1 expression dynamics in the saccule
(see Fig. S2 in the supplementary material), although contraction occurred
largely along the anteroposterior axis of this structure, rather than along
the medial-lateral axis as in the utricle. Importantly, all tamoxifen start
times tested (ranging from E8.5-12.5) resulted in a cohort of macular Ngn1
derivatives comprising both hair cells and pseudostratified epithelial cells.
These data, together with results described in the previous section, indicate
that macular sensory cells can derive from cells that express Ngn1 at
any time between E9 and E14.

Math1 suppresses neurogenesis in the developing utricle and
saccule

Our results indicate that the domain of Ngn1+ precursor
cells is gradually transformed from a purely neurogenic region into sensory
epithelia of the utricular and saccular maculae. Since functional antagonism
between related bHLH transcription factors has been described in other systems
(Fode et al., 2000;
Gowan et al., 2001;
Akagi et al., 2004), we tested
whether Math1 (which is required for sensory epithelial differentiation)
(Bermingham et al., 1999)
suppresses neurogenesis by inhibiting Ngn1 transcription in otic
epithelial cells. We found that Math1 function is required for the
normal contraction of epithelial Ngn1 expression characterized in the
previous section (Fig. 4A,B;
data not shown). We quantified expression of the Ngn1-GFP reporter and
NeuroD in Math1-null homozygote embryos and observed large
(>6×) increases over wild type in the numbers of neural precursors
within the developing utricle and saccule; a less severe form of this
phenotype was found in Math1 heterozygotes
(Table 1). Excess neural
precursors were seen to delaminate and migrate away from the mutant epithelium
to form an VIIIth cranial ganglion that was larger than wild type (see Fig.
S3B asterisk, E-H, in the supplementary material). Ectopic neurogenesis in
Math1-/- epithelia localized specifically to parts of the
utricle and saccule that normally differentiate as sensory maculae, and was
not detected in the cristae, cochlea, or any non-sensory epithelia
(Fig. 4D,F; see Fig. S3A-D in
the supplementary material; data not shown); in Math1 heterozygotes,
it occurred only at the interface of neurogenic and sensory regions
(Fig. 4E, bracket). In
Math1-/- epithelia, marked excess neurogenesis at E14.5
and E15.5 followed a partial decline in neurogenic activity through E13.5
(Fig. 4N). This initial
declining trend in neurogenic activity in mutants, which is similar to that of
wild type, suggests that factors in addition to Math1 contribute to
early neurogenic suppression.

Ngn1 loss-of-function causes a failure of neural precursor
generation in the otic epithelium and absence of the VIIIth cranial ganglion
(Ma et al., 1998;
Ma et al., 2000). If
antagonism between Math1 and Ngn1 during ear development is
reciprocal, then Ngn1 loss-of-function should result in ectopic hair
cells. Our analysis of sensory marker expression in the
Ngn1-/- utricle confirmed this prediction. As indicated by
both Math1 and myosin VIIa expression, the
Ngn1-/- utricular macula at E13.5 and E14.5 was expanded
medially into the region that is normally neurogenic
(Fig. 4C,F,H,K,M). At E13.5,
the total area of Math1 expression in the Ngn1-/-
utricle exceeded that of wild type by 39% (P<0.002, n=3
epithelia per genotype), and there were more Math1+ cells
in the Ngn1-/- utricle than in wild type at E13.5 and
E14.5 (Table 1). Growth
abnormalities of the Ngn1-/- mutant ear
(Matei et al., 2005;
Ma et al., 2000) confounded
our analysis of other structures. The saccule was profoundly hypoplastic (see
Fig. S4A in the supplementary material), and we correlated this to a period of
intense, region-specific apoptosis in the otic epithelium between E11.5 and
E12.5 (see Fig. S4B,C in the supplementary material; data not shown).
Ngn1-/- cristae were also smaller than their wild-type
counterparts, which was evident from our quantification of
Math1+ cells in the Ngn1-null mutant lateral
crista (Table 1).

Ngn1 expression decreases over time in the prospective
utricle. (A) E14.5 utricular macula schematized in the same
orientation as the scatterplots in B and E-E″. A cross-section of the
macula along the dotted line is represented at the bottom. The macula (region
of myosin VIIa staining) is in gray. The active neurogenic region (Ng, region
of Ngn1-GFP staining) is white. ut m-lc, non-sensory tissue between the
utricular macula and lateral crista; ut m-sac m, non-sensory tissue between
the utricular macula and saccular macula. (B) Distribution of
GFP+ cells (blue crosses, cyan in image) from three E14.5 Ngn1-GFP
BAC transgenic utricles, plotted on a normalized scale, defines the region of
active neurogenesis. Gray area represents an averaged macular area (myosin
VIIa +); the yellow area is non-sensory epithelium between the
utricular macula and lateral crista. White arrow marks the lateral extent of
the macula. (C) Number of Ngn1 derivatives in the Ngn1-CreER;Z/EG
utricle versus time of first tamoxifen feeding for embryos sacrificed at
E14.5. Each point is based on four or more ears from two or more litters.
(D) Sections through utricular maculae, representing the change in the
spatial distribution of Ngn1 derivatives (green) with different tamoxifen
regimens. Downturned brackets indicate overlap between Ngn1 derivatives and
myosin VIIa + hair cells. Upturned brackets indicate the region of
active neurogenesis. White arrows indicate the lateral extent of the macula.
Asterisk shows an Ngn1 derivative in non-sensory tissue between the utricular
macula and lateral crista. (E-E‴) Spatial distributions of Ngn1
derivatives from E14.5 utricles exposed to different tamoxifen administration
regimens, as indicated at the top of each plot. (E) n=4 utricles;
(E′) n=6 utricles; (E″) n=14 utricles. Ngn1
derivative cell types and regions of the plot are coded as shown in
E‴.

The inner ear of Ngn1 heterozygotes was not grossly dysmorphic and
therefore provided a context for analyzing cell fate changes in response to
reduced Ngn1 levels. We quantified Math1+ cells
in the utricle, saccule and lateral crista of Ngn1 heterozygote and
wild-type littermates. In the Ngn1+/- utricle and saccule,
numbers of Math1+ cells were increased by 16-29% compared
with wild type (Table 1). This
was due to an expansion in the area of Math1+ macular
domains in heterozygotes (Fig.
4K,L) and an increased density of Math1+ cells
within heterozygote maculae compared with wild type
(Fig. 4O). Unlike the maculae,
the Ngn1+/- lateral crista - a structure that does not
express Ngn1 during normal development - showed no increase in
Math1+ cells compared with wild type
(Table 1). These results
suggest that the numbers of Math1+ cells are increased in
response to Ngn1 hemizygosity specifically at sites where the two
genes are co-expressed (utricle and saccule).

Although, as described above, myosin VIIa expression domains are expanded
in Ngn1 mutants (Fig.
4F-H), we found fewer myosin VIIa+ cells in the
Ngn1-/- utricle and Ngn1+/- utricle
and saccule than in wild type (Table
1). This was owing to a lower density of myosin VIIa+
cells in the mutants as compared with wild type and, in addition, myosin
VIIa+ hair cell size and shape were abnormal in the mutants (see
Fig. S4D-F in the supplementary material). Neither TUNEL, nor anti-caspase 3
immunohistochemistry, at E14.5 provided evidence that these abnormalities were
due to apoptosis (data not shown). Since the onset of myosin VIIa expression
normally follows Math1 expression, and all of the mutant sites under
consideration have more Math1+ cells than wild type, a
considerable number of Math1+ macular cells in
Ngn1 mutants (greater than 35%) must fail to express myosin
VIIa+. Taken together, our results indicate that at sites of
Ngn1 and Math1 co-expression (utricle and saccule), reduced
Ngn1 gene dose causes excess numbers of Math1+
cells to form, but many of these cells do not properly differentiate as hair
cells.

Ngn1 negatively regulates its own expression and is
inhibited by Notch signaling

Ngn1 is thought to function analogously to the Drosophila
proneural genes (Ma et al.,
1996; Ma et al.,
1998; Cornell and Eisen,
2002). Consistent with a proneural function for Ngn1, we
found that Ngn1-GFP signal varies in intensity between neighboring cells, and
is strongest in delaminating cells (Fig.
5A). We therefore tested whether Ngn1 negatively
regulates its own transcription in the otic epithelium by comparing expression
patterns of the Ngn1-GFP BAC transgene in Ngn1-null homozygote and
wild-type littermates prior to the appearance of ectopic Math1
expression. At E12.5, Ngn1-GFP is normally expressed in a speckled pattern by
a subset of cells in the neurogenic region
(Fig. 5B). By contrast, in
Ngn1-/- embryos, the GFP signal was present in all cells
of the region, with little variability in signal strength between cells
(Fig. 5D). Loss of the speckled
expression pattern for the Ngn1-GFP transgene did not occur on a
Math1-/- background
(Fig. 5C), indicating that this
phenotype is specific to the loss of Ngn1. We also found excess
Ngn1-GFP+ and NeuroD+ epithelial cells in the
Ngn1+/- utricle and saccule compared with wild type
(Table 1). In
Ngn1-/- ears, expression of the Ngn1-GFP transgene was
completely abolished by E14.5, presumably owing to inhibition by ectopic
Math1 expression (Fig.
4H,M).

To investigate potential interactions of Ngn1 with Notch
signaling, we assayed expression of the Notch ligand Dll1 in the
Ngn1-null homozygote. We found reduced Dll1 expression in
the primitive utricle and saccule of mutants as compared with wild type
(Fig. 5E,F) (see also
Ma et al., 1998). To test
whether Notch activity suppresses Ngn1 transcription within the otic
epithelium, we analyzed Ngn1 expression in embryos lacking
Pofut1. This gene encodes the protein O-fucosyltransferase 1, which
glycosylates epidermal growth factor-like repeats within the extracellular
domain of the Notch receptor (Lei et al.,
2003; Okajima et al.,
2003; Okajima et al.,
2005). Pofut1 loss-of-function abolishes ligand-induced
Notch signaling and causes phenotypes similar to those of embryos lacking
downstream effectors of all Notch receptors, including mid-embryonic lethality
(Shi and Stanley, 2003). We
therefore assayed Ngn1 expression in early Pofut1 embryos
(E9-9.5), when the otic placode invaginates to first form an otocyst. At these
stages, Ngn1 mRNA signals were increased in
Pofut1-/- embryos as compared with wild-type littermates
in the otic epithelium, midbrain, trigeminal placode, epibranchial placodes
and spinal cord (Fig. 5G,H),
indicating that Ngn1 transcription in all these embryonic regions is
negatively regulated by canonical Notch signaling. Thus, the negative
autoregulation of Ngn1 in the otic epithelium might be controlled by
Notch-mediated lateral inhibition.

Positive autoregulation of Math1 in the inner ear
epithelium

Math1 positively autoregulates its transcription at particular
sites in the embryo (Helms et al.,
2000). To determine whether Math1 is subject to positive
autoregulation during ear development, transgenic reporters of Math1
promoter activity were compared across wild-type and
Math1-/- backgrounds. A BAC that expresses GFP under the
control of Math1 regulatory elements mimics patterns of
Math1 mRNA expression in the developing ear
(Fig. 6A), hindbrain region
(Fig. 6A, inset), spinal cord,
and other sites in the embryo (J.E.J., unpublished). By contrast, we were
unable to detect GFP signal in the ears of
Math1-/-::Math1-GFP BAC embryos at stages E13.5 through
E15.5 (Fig. 6B; data not
shown), although GFP expression was clearly present at other sites of
expression, such as the hindbrain (Fig.
6B, inset). A second transgenic line, which carries a 1.4 kb
Math1 enhancer with an E-box site that is essential for Math1 binding
and autoregulation in other tissues (Helms
et al., 2000), also mimics Math1 expression in the
developing wild-type ear (Chen et al.,
2002; Lumpkin et al.,
2003). As with the Math1-GFP BAC, we found no GFP reporter signal
in the sensory epithelia of these Math1-/-::Math1-GFP
embryos at stages E13.5 through E15.5 (Fig.
6C,D). The complete lack of reporter expression in the cristae of
both Math1-/-::Math1-GFP reporter lines indicates that the
phenotype is not due solely to inhibition by ectopic expression of
Ngn1.

DISCUSSION

The neurogenic region of the otocyst gives rise to sensory epithelia
of the utricle and saccule

It has been unclear how neurogenic tissue of the otocyst relates to sensory
epithelia of the inner ear. Gene expression studies in both chicken and mouse
suggest that neurogenesis occupies only part of a larger sensory-competent
domain of the otocyst (Morsli et al.,
1998; Adam et al.,
1998; Cole et al.,
2000; Fekete and Wu,
2002), and a recent lineage study in the chicken shows that
vestibular and spiral (auditory) neurons of the VIIIth ganglion can be
clonally related to utricular epithelial cells (both macular and non-sensory)
(Satoh and Fekete, 2005). By
fate mapping in mouse, we show that Ngn1+ otic epithelial
cells can differentiate as vestibular or spiral ganglion neurons, hair or
supporting cells of the utricular and saccular maculae, or non-sensory
epithelial cells surrounding the maculae. Although the question remains open
as to whether, in mouse, these cell types descend clonally from a common
progenitor, our work unambiguously traces the origin of specific sensory
(macular) and non-sensory cells to the neurogenic domain of the otocyst.

Our fate mapping suggests that the other functional class of vestibular
sensory epithelia, the cristae, and their associated semicircular canals do
not derive from the Ngn1+ domain of the otocyst. This
confirms previous gene expression studies tracing the origin of cristae to a
Bmp4+ region outside of, and adjacent to, the neurogenic
domain (Morsli et al., 1998;
Raft et al., 2004)
(Fig. 7A′). The very rare
occurrence (a few cells in one of 20 ears) of Ngn1 derivatives in the lateral
crista suggests that mixing of cells between the neurogenic and Bmp4
domains occurs infrequently. Whether this lack of mixing is due to
differential affinity between the two regions, or whether neurogenesis is
actively suppressed within the Bmp4 domain, is not clear. In support
of the latter hypothesis, Tbx1, a T-box gene that inhibits
Ngn1 and maintains Bmp4 expression in the otocyst
epithelium, is expressed continuously and from very early stages in the
presumptive and definitive cristae (Arnold
et al., 2006; Raft et al.,
2004; Vitelli et al.,
2003).

Our mapping of Ngn1-GFP and NeuroD expression domains revealed no
evidence of active neurogenesis in the definitive cochlea. However, we did
find Ngn1 derivatives in a non-sensory region of the cochlea (the GER) in the
majority of ears analyzed. Based on its location in the ear, the GER might
derive from the most posteroventral-medial edge of the otocyst neurogenic
region. Interestingly, the GER lies immediately adjacent to the organ of
Corti, within which we found no Ngn1 derivatives. This result, the common
occurrence of Ngn1 derivatives in non-sensory tissue between the utricula
macula and the anterior/lateral cristae (but not in the cristae)
(Fig. 2E,G), and the initiation
of macular Math1 expression as stripes just within opposite borders
of the neurogenic domain, support the hypothesis that sensory epithelia are
induced at or near compartment boundaries in the otocyst
(Fekete, 1996;
Brigande et al., 2000).

Cross-inhibition between Math1 and Ngn1 segregates
a progenitor field of dual competence into distinct neurogenic and sensory
cell populations

We show that neurogenesis and hair cell production, long considered
strictly sequential, actually overlap in the developing utricle and saccule
for several days of gestation. During this period, neural precursors and
nascent hair cells initially intermingle and later sort out across
well-defined borders. Functionally, we show that Math1 and
Ngn1 mutants have complementary inner ear phenotypes, supporting the
hypothesis that mutual antagonism between these genes coordinates neurogenesis
and hair cell production (Matei et al.,
2005). Loss of Math1, which is normally expressed in all
sensory regions of the ear, leads to excess and ectopic neurogenesis only in
sensory regions with a history of Ngn1 expression (utricular and
saccular maculae). This effect is gene dose-sensitive, as Math1
heterozygotes exhibit a neurogenic phenotype intermediate to those of the
Math1-null homozygote and wild type. Conversely, Ngn1
hemizygosity causes excess and ectopic Math1 expression specifically
in the utricle and saccule, and although Ngn1-null homozygosity
causes growth abnormalities of the ear, the Ngn1-/-
utricle still shows a phenotype of excess and ectopic Math1
expression. These effects are seen only at sites in the developing ear where
Ngn1 and Math1 are co-expressed, and we propose that they
result from a disruption of close-range cross-inhibition. Cross-inhibition
might influence multiple steps in the process, whereby an
Ngn1+ progenitor field of dual competence (neural and
sensory epithelial) is gradually restricted to producing only sensory
epithelial cells. These include: (1) Math1 domain establishment
within opposite borders of the Ngn1+ region
(Fig. 7A′); (2)
Math1 domain expansion and decline in Ngn1 expression
(Fig. 7A′,A″); and
(3) compartmentalization of the region into a pair of adjacent Math1
(sensory) and Ngn1 (neurogenic) domains
(Fig. 7A″). The potential
basis for the competitive advantage of Math1 over Ngn1 in
this system is discussed below.

Ngn1, but not Math1, functions as a proneural gene
during mouse ear development

Criteria for proneural function include early, broad expression of
transcript in all cells of a germinal epithelium and subsequent refinement of
transcription to a subset of cells by lateral inhibition
(Jan and Jan, 1993;
Lewis, 1996). We find no
evidence of these features in our studies of Math1 expression in the
vestibular system of the mouse. Of the two genes relevant to this study, it is
Ngn1 and not Math1 that initially marks the prospective
maculae and exhibits the variegated expression among neighboring cells that is
characteristic of proneural genes (Fig.
5A,B). Furthermore, using two different transgenic reporter lines,
we show that Math1 is required for detectable levels of
Math1 reporter expression in the otic epithelium, suggesting that
Math1 promoter activity is amplified and maintained by positive
autoregulation. One possible consequence of this is a rapid and irreversible
commitment of progenitors to the hair-cell fate once Math1
transcription surpasses a threshold for positive autoregulation. Our results
thus support the view that Math1 functions as a hair-cell commitment
factor rather than a proneural (or `prosensory') gene
(Chen et al., 2002) (for a
review, see Kelley, 2006).
Interestingly, in zebrafish, which has two atoh1 genes, differences
in the timing and autoregulation of Math1/atoh1 genes from
that described here lead to the opposite conclusion
(Millimaki et al., 2007). For
example, zebrafish atoh1a and 1b are required for hair cell
generation, but their expression precedes that of ngn1
(Andermann et al., 2002) and
marks the prospective maculae from very early stages. Gene duplication and
evolutionary pressure on the regulatory genome might therefore dictate the
precise functions of Math1/atoh1 during ear development in
different species.

Our experiments reveal a profile of Ngn1 autoregulation very
different from that of Math1. We demonstrate that Ngn1 is
required to limit its own transcription within the otic epithelium. We also
extend a previous observation that proper otic expression of the Notch ligand
Dll1 is dependent on Ngn1
(Ma et al., 1998) and show a
pattern of increased Ngn1 expression in the early otic epithelium of
Pofut1-/- embryos, which are deficient in canonical Notch
signaling (Shi and Stanley,
2003). These results and the expression of Ngn1 in neural
and sensory progenitors from very early stages fulfill several criteria for
proneural function. Likely consequences of the proneural activity of
Ngn1 are control over the pace of neurogenesis during otocyst stages
and preservation of an uncommitted progenitor cell population for sensory
development. These functions are consistent with recently reported effects of
conditional Dll1 loss-of-function in the developing ear, which
include an enlarged ganglion rudiment and specific hypoplasia of the utricle
and saccule (Brooker et al.,
2006), and with blockade of Notch signaling in the chicken, which
causes excess neurogenesis at the expense of sensory epithelial precursors
(Daudet et al., 2007).

Given our evidence for a mutual antagonism between Ngn1 and
Math1, how does Math1 exert the stronger inhibitory activity
so that sensory epithelia replace an active neurogenic region? Our model
states that Ngn1 promotes a neural fate cell-autonomously and keeps
its own expression low or off in neighboring cells through Notch-mediated
lateral inhibition (Fig. 7B,C).
Cells expressing high levels of Ngn1 delaminate from the epithelium
as neural precursors. Cells remaining within the neurogenic epithelium
constitute a dynamic mix of committed neural precursors and uncommitted
progenitors. The latter group may adopt a neural fate in subsequent rounds of
delamination or may remain uncommitted for several days, after which they
adopt hair or supporting cell fates in response to Math1 induction
within the region (Fig.
7B′,B″,C′). This is supported by our fate
mapping results, as Ngn1 derivatives can have any of these identities. Once
Math1 transcription exceeds a particular threshold, positive
autoregulation irreversibly commits progenitors to a hair cell fate, and
committed hair cells may then induce the supporting cell phenotype through
intercellular signaling (Woods et al.,
2004). Since strongly Ngn1+ cells continuously
delaminate from the epithelium, Math1-expressing cells are left to
interact with epithelial progenitors expressing lower levels of Ngn1.
These features might bias the mutual antagonism between Ngn1 and
Math1, thereby promoting sensory epithelial differentiation at the
expense of continued neurogenesis.

Conclusion

We have implicated cross-regulation between bHLH genes and differential
autoregulation as mechanisms for converting a neurogenic epithelium into
specialized mechanosensory receptors. A novel aspect of this work - and one
that is potentially relevant to other systems - is the dynamic nature of the
patterning processes described. We show that progressive regionalization of
bHLH genes through cross-inhibition can result in a sequential and overlapping
production of distinct cell types, and that differential autoregulation might
provide the driving force for such a transition.

Many questions remain unanswered. For example, does cross-inhibition
between Ngn1 and Math1 occur within a single cell, through
intercellular signaling, or by a combination of these two mechanisms?
Cell-autonomous cross-inhibition might convert a weakly
Ngn1+ cell directly into a Math1+
nascent hair cell. Alternatively, if the antagonism occurs through
intercellular signaling, Ngn1+ cells might pass through a
`sensory-restricted progenitor' state before committing to the hair-cell fate
(Fig. 7C′). We find the
latter alternative attractive given that embryonic maculae contain many Ngn1
derivatives with a pseudostratified epithelial (non-hair-cell) phenotype.
Molecular and cellular mechanisms underlying the apparent compartmentalization
of sensory and neurogenic regions also warrant scrutiny, as there is abundant
evidence that Notch-mediated intercellular signaling occurs at nascent
boundaries during development (Irvine,
1999). In summary, our results form a basis for understanding how
progenitors are allocated to various cell fates during inner ear
development.

Daudet, N., Ariza-McNaughton, L. and Lewis, J.
(2007). Notch signaling is needed to maintain, but not to
initiate, the formation of prosensory patches in the chick inner ear.
Development134,2369
-2378.

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